Capillary electrophoresis is one of the most widely used separation techniques in the biologically-related sciences. Capillary electrophoresis is a technique which permits rapid and efficient separations of charged substances. In general, capillary electrophoresis involves introduction of a sample into a capillary tube, and the application of an electric field to the tube. The electric potential of the field both pulls the sample through the tube and separates it into its constituent parts, or separates the components of the sample based upon the relative sizes of those components. An on-line detector can be used to continuously monitor the separation and provide data as to the various constituents. Capillary electrophoresis can be generally separated into two categories based upon the separating medium, these being "open tube" and "gel" capillary electrophoresis.
In "open tube" capillary electrophoresis, the capillary tube is filled with an electrically conductive buffer solution. Upon ionization of the capillary, the negatively charged inner wall will attract a layer of positive ions from the buffer. As these ions flow towards the cathode, under the influence of the electrical potential, the bulk solution (i.e., the buffer solution and the sample being analyzed), must also flow in this direction to maintain electroneutrality. This electroendosmotic flow provides a fixed velocity component which drives both neutral species and ionic species, regardless of charge, towards the cathode. Separations utilizing open tube capillary electrophoresis typically rely upon the charge-to-mass ratios of the constituents traversing the column. I.e., those constituents having a (relative) high charge-to-mass ratio typically transit the column faster than constituents having a lower charge-to-mass ratio.
In "gel" capillary electrophoresis, the capillary is filled with an appropriate separation gel composition. Molecular species such as proteins, peptides, nucleic acids, and oligonucleotides can be separated by causing the species to migrate in a buffer solution under the influence of an electric field. The buffer solution normally is used in conjunction with a low to moderate concentration of the gel composition, such as agarose or polyacrylamide, which functions to minimize the occurrence of mixing of the species being separated. Two primary separating mechanisms exist: a) separations based on differences in the effective charge of the species; and b) separations based on molecular size.
The first of these mechanisms is generally limited to low or moderate molecular weight materials, such as small oligonucleotides (about 1 to about 50 nucleotides in length). This is because there is typically an insignificant difference between the effective charges of high molecular weight materials, making the task of separation difficult or impossible.
Separations based on molecular size are generally referred to as molecular "sieving". Molecular sieving utilizes gel matrices having controlled pore sizes as the separating medium. The separation results from the relative abilities of the different size molecular species to penetrate through the gel matrix; smaller molecules move more quickly than larger molecules through a gel of a given pore size.
Medium-to-high molecular weight oligonucleotides (greater than about 50 nucleotides in length), polypeptides, and proteins are commonly separated by molecular sieving electrophoresis. Proteins comprise both negative charged and positive charged moieties. As such, proteins become charged molecules as they transit a capillary column under the influence of an electric field. Accordingly, in order to separate proteinaceous materials based upon the size of the molecules, these materials must have the same effective charge to mass ratio as they traverse the capillary column.
Achieving the same effective charge to mass ratio is commonly accomplished by treating the proteinaceous materials with a surfactant, such as sodium dodecyl sulphate ("SDS"), and utilizing a polyacrylamide gel material as the sieving medium. Such a procedure is referred to as sodium dodecyl sulphate polyacrylamide gel electrophoresis ("SDS-PAGE"). See, for example, Gel Electrophoresis of Proteins: A Practiced Approach (Second Ed). B. D. Harnes & D. Rickwood, Eds. IRL Press, Oxford University Press, 1990. See also, New Directions in Electrophoretic Methods, T. W. Jorgenson & M. Phillips, Eds. published by American Chemical Society, Washington, D.C. 1987. Both of these references are incorporated fully herein by reference.
A surfactant, such as SDS, comprises a hydrophobic (water-hating) "tail" and a hydrophilic (water-loving) "head." Thus, a surfactant interacts with a protein species via hydrophobic interactions between the hydrophobic "tail" of the surfactant and the protein species. Upon ionization, the hydrophilic "head" of the surfactant molecules surrounding the protein species become negatively charged, positively charged, or remain neutral; upon ionization, SDS becomes negatively charged. Accordingly, an SDS:protein complex has a uniform charge distribution, and such a complex can then be separated based upon size relative to the pore-size distribution throughout the gel matrix.
Commercially available capillary electrophoresis instruments, such as the P/ACE.TM. high performance capillary electrophoresis system (Beckman Instruments, Inc., Fullerton, Calif., U.S.A.), utilize a detection system based upon ultra-violet ("UV") light absorption. While UV detection of SDS-protein complexes in polyacrylamide filled capillaries is possible, such detection is limited to a specific wavelength detection of about 250 nm and higher. This is because of the high UV absorbance associated with both crosslinked and uncross-linked polyacrylamide gels.
Such detection limitations are a distinct disadvantage particularly with respect to the analysis of proteins. This is because proteins absorb UV light very strongly at 214 nm, due to peptide bonds within proteins. Thus, UV detection of proteins should be conducted at about 214 nm. However, because of the 250 nm and higher detection limitations created by the use of polyacrylamide gels, the sensitivity and selectivity of UV detection of proteinaceous materials using polyacrylamide-based gel systems is limited.
Accordingly, UV detection of surfactant: proteinaceous materials would be greatly improved if on column detection was conducted at lower UV wavelengths. This, in light of the foregoing, requires molecular sieving materials that do not suffer the drawbacks of polyacrylamide gels.
Unlike proteins and peptides, polynucleotides (i.e., macromolecules of deoxyribonucleic acid, DNA, and ribonucleic acid, RNA) have the same charge-to-mass ratio. As such, the analysis of, e.g., DNA, does not typically require utilization of a surfactant as described above. Most typically, the analysis of polynucleotides involves a determination of the sequence thereof, i.e., the determined order of specific nucleic acids along the polynucleotide, or the analysis of restriction fragments, e.g., the analysis, by length, of inherited genetic variations based upon the comparative fragment sizes of polynucleotides subjected to enzymatic cleavage (referred to as "restriction fragment length polymorphisms", or "RFLPs"). Such sequence information provides a wealth of knowledge to research, commercial and medical investigators. RFLPs are utilized, e.g., to correlate the appearance of particular genetic variations with particular polynucleotide fragment lengths.
DNA and RNA are long, threadlike macromolecules, DNA comprising a chain of deoxyribonucleotides, and RNA comprising a chain of ribonucleotides. A nucleotide consists of a nucleoside and one or more phosphate groups; a nucleoside consists of a nitrogenous base linked to a pentose sugar. Typically, the phosphate group is attached to the fifth-carbon ("C-5") hydroxyl group ("OH") of the pentose sugar. Accordingly, such compounds are typically referred to as nucleoside 5'-phosphates or 5'-nucleotides.
In a molecule of DNA, the pentose sugar is deoxyribose, while in a molecule of RNA, the pentose sugar is ribose. The nitrogenous bases in DNA can be adenine ("A"), cytosine ("C"). guanine ("G"), or thymine ("T"). These bases are the same for RNA, except that uracil ("U") replaces thymine. Accordingly, the major nucleotides of DNA, collectively referred to as "deoxynucleotide triphosphates" ("dNTPs"), are as follows: deoxyadenosine 5'-triphosphate ("dATP"); deoxycytidine 5'-triphosphate ("dCTP"); deoxyguanosine 5'-triphosphate ("dGTP"); and deoxythymidine 5'-triphosphate ("dTTP"). The major nucleotides of RNA are as follows: adenosine 5'-triphosphate ("ATP"); cytidine 5'-triphosphate ("CTP"); guanosine 5 '-triphosphate ("GTP"); and uridine 5'-triphosphate ("UTP"). By convention, the base sequence of nucleotide chains is written in a 5' to 3' direction, i e , 5'-ATCG-3', or simply ATCG.
Two complementary single chains (or "strands") of nucleotides, held together by (relatively) weak hydrogen bonds between the nucleotides, form a complete double-stranded DNA or RNA macromolecule. The specificity of binding between the bases is such that A always binds to T (or U in the case of RNA), and C always bonds with G. Thus, for the sequence 5'-ATCG-3', the sequence 3'-TAGC-5' will lie immediately across therefrom. Because of this specificity in binding, the sequence of a single-stranded template of DNA or RNA can be determined by determining the bases which bind to the template. This, in essence, is the basis for nucleotide sequencing.
Another unique and useful form of NTPs exist. These are referred to as chain terminating dideoxynucleotide triphosphates, or "ddNTPs." ddNTPs differ from dNTPS in that they lack a 3'-hydroxyl group. Accordingly, while ddNTPs can be incorporated into the growing primer strand via the 5'-triphosphate portion thereof, the absence of a 3'-hydroxyl group prevents formation of a phosphodiester bond with a succeeding dNTP (or ddNTP). Accordingly, once a ddNTP is incorporated into the primer strand, further extension of that strand is not possible.
DNA sequencing protocols, particularly those suited for automated DNA sequencing instrumentation formats, principally rely upon the methodology developed by Sanger et al., Proc. Natl. Acad. Sci. USA 74:5463-5467 (1977) (hereinafter, "Sanger et al."). Generally, the Sanger et al. protocol involves four separate syntheses, whereby a single stranded template (i.e., the sequence to be determined which can be obtained via, e.g., denaturation of double-stranded DNA or cloning of the DNA template into, e.g., bacteriophage M13 vector), is provided with a primer (i.e., a short oligonucleotide complementary to a portion of the template) such that elongation of the primer proceeds via DNA polymerase (an enzyme that brings about the incorporation of a dNTP or ddNTP along the growing primer strand). Each reaction is terminated (via the appropriate ddNTP utilized in that reaction) at one of the four bases, i.e., A, T, C, or G, via the incorporation of the appropriate chain terminating agent. Thus, if the templates have the sequence 5'-XXXATGCTGCA-3' and the primer is complementary to XXX, the addition of dGTP, dCTP, dTTP, dATP and ddATP, as well as DNA polymerase, would lead to the formation of two primer-extension fragments: 5'-XXXTA-3' and 5'-XXXTACGA-3', which are complementary to the template. In a second synthesis, the protocol would be the same, except that, e.g., ddTTP would be utilized instead of ddATP, leading to the formation of 5'-XXXT-3' and 5'-XXXTACGACGT-3'. The third synthesis would utilize ddCTP (5'-XXXTAC-3' and 5'-XXXTACGAC-3'), and the fourth would utilize ddGTP (5'-XXXTACG-3' and 5'-XXXTACGACG-3'). By utilizing labelled ddNTPs, dNTPs or primer, and subjecting the various extension products to gel electrophoresis, various discrete bands will be obtained on the resulting gel, due to the various electrophoretic mobilities of the extension fragments. From these bands, one can determine the sequence of the extension fragments, such that the sequence of the template is readily determined therefrom.
While polynucleotides have the same charge-to-mass ratio, single stranded polynucleotides, necessary for sequencing reactions, suffer from the possibility of secondary structure formation, i.e., the folding of the single strand via internal base-pairing of complementary bases along the single strand. Thus, the sequencing gel typically incorporates a denaturing material therein, i.e., a material that reduces or prevents such secondary structure formation.
As can be appreciated from the foregoing, different separating materials can be utilized for different protocols. Typically, however, this requires the use of different capillary columns for each protocol. This is because such separating materials (other than the buffers used in open tube capillary electrophoresis) can become substantially "fixed" within the column. Thus, it has, heretofore, been impractical to attempt to remove the separation material from the column for any reason such that each time a new material is required for separating samples, an entirely new column must be obtained or prepared. Such columns are typically expensive, the preparation thereof is tedious and time consuming, and replacement thereof requires the shut-down of the capillary electrophoretic instrument, thus decreasing throughput and increasing the time for analysis.
What are needed, then, are capillary columns useful in the analysis of a variety of materials, and which are compatible with different separating materials, such that a separating material used for sample analysis can be removed from the column, and a different separating material can be added thereto, without affecting the performance of the column or any coatings utilized therewith.